Enteroviruses constitute a broad range of pathogens etiologically responsible for a wide range of diseases in humans, as well as in other animals. The genus Enterovirus is a member of the family Picornaviridae. As the family name indicates, enteroviruses are small RNA viruses; they contain positive single stranded RNA as the genome. Five groups are found within the enteroviruses: coxsackievirus A (CA), coxsackievirus B (CB), echovirus (E), and numbered enteroviruses (EV), as well as poliovirus (PV). There are 66 serotypes currently classified among the human enteroviruses, although two serotypes, E22 and E23, are to be reclassified in a different genus.
The viral genome is shown schematically in FIG. 1. The single stranded RNA comprises a 5′ nontranslated region (single line), which is followed by an open reading frame coding for a polyprotein precursor of Mr 240-250×103 Da (boxed portion), followed by a 3′ noncoding sequence and a poly (A) tract (single line). In the polyprotein, the sequence of gene products begins 1A, 1B, 1C, 1D, and 2A. 1A through 1D are, respectively, the structural proteins VP4, VP2, VP3, and VP1 of the viral capsid; VP1 is followed in the open reading frame by a nonstructural protein 2A.
The various members of the human enteroviruses cause a wide range of symptoms, syndromes and diseases. These include acute benign pericarditis, acute flaccid paralysis, acute hemorrhagic conjunctivitis, aseptic meningitis, various exanthemas, carditis, croup, encephalitis, enanthema, gastrointestinal disease, hepatitis, hand-foot-and-mouth disease, various respiratory diseases, myocarditis, neonatal disease including multi-organ failure, pericarditis, pleurodynia, rash, and undifferentiated fever. In general, the syndromes are not correlated with particular enterovirus serotypes, nor does a serotype specifically correlate with a particular disease, although in certain cases serotypes do correlate with particular diseases.
Enteroviruses are responsible for large numbers of infections. There may be between 30 million to 50 million illnesses that are ascribable to enteroviruses each year in the United States (CDC; MMWR 46:748-750; Strikas et al. J. Infect. Dis. 146:346-351 (1986); Rotbart in Human Enterovims Infections, H. A. Rotbart (ed.) ASM Press, Washington, D.C., pp. 401-418 (1995)). After rhinoviruses, enteroviruses are the most common viral infection in humans. Enteroviral infections lead to 30,000 to 50,000 hospitalizations each year for aseptic meningitis, myocarditis, encephalitis, acute hemorrhagic conjunctivitis, nonspecific febrile illnesses, and upper respiratory infections (Melnick, Biologicals 21:305-309 (1993); Morens et al. in Human Enterovirus Infections, H. A. Rotbart (ed.) ASM Press, Washington, D.C., pp. 3-23 (1995); Melnick in Fields Virology (B. N. Fields et al. (eds.) 3rd ed., Lippincott-Raven Publishers, Philadelphia, pp. 655-712 (1996)). Enteroviruses are also implicated in acute flaccid paralysis in animal models, as well as in dilated cardiomyopathy. The six serotypes of coxsackie B viruses are implicated in a variety of clinical diseases, such as meningitis, myocarditis and severe neonatal disease. Recently, enterovirus infection has been linked to chronic fatigue syndrome (Elements et al., J. Med. Virol. 45:156-161 (1995)).
Poliovirus is also an enterovirus that infects humans. Three serotypes, PV1, PV2, and PV3 are known. A nonenteroviral picornavirus that also afflicts humans is human rhinovirus (HRV), responsible for many common cold infections; several serotypes have been identified. Additionally, picornaviruses affect mammals other than humans, including viruses such as bovine enterovirus (BEV) and simian picornavirus (SPV).
It is important to identify the serotype of an enterovirus infection in a subject. Knowledge of the serotype can provide useful guidance to a physician in determining a course of treatment of the disease in the subject. For example, the appropriately identified immune globulin having a sufficient titer may be administered to immunocompromised patients. Furthermore, an antiviral drug such as Pleconaril (Viropharma) may differ in its relative efficacy against different serotypes. Additionally, an understanding of the geographic and chronological development of an enterovirus infection in a population can influence preventive measures among the members of the population to minimize the spread of the disease. Furthermore, it is useful from a broader perspective to track the incidence and distribution of an enterovirus disease from an epidemiological point of view. In earlier practice, it was to found that the various serotypes could be grown in different cell culture hosts, and in different animal model hosts. In the animal hosts, furthermore, different symptomology also provided typing information. These classical assays provide ways of distinguishing the serotypes. Nevertheless, some enterovirus serotypes, especially in the coxsackievims A group, do not grow in cell culture. It has been observed that 25% to 35% of patient specimens are not identified by cell culture for a variety of reasons (Rotbart, 1995). Furthermore, such culturing and classification procedures are costly, time-consuming, subject to experimental variation, and not amenable to repetitive or extensive application in the field.
The serotypes of non-polio enteroviruses have been identified during the past several decades using classical immunological neutralization assays based on a panel of specific antibodies. Application of such a determination to a clinical sample is generally impractical and inconvenient. Although a number of neutralization sites have been localized to the VP1 protein of enteroviral particles, the exact identity of the epitopes responsible for serotype specificity remain unknown; VP2 and VP3 may also contain specific neutralizing epitopes. Serotyping has generally been carried out using intersecting pools of antisera, the Lim and Benyesh-Melnick (LBM) pools, which were originally defined in 1960 (Lim et al., J. Immunol. 84:309-317 (1960)). The antiserum pools currently distributed by the World Health Organization cover 42 serotypes in 8 pools (Melnick et al., Bull. WHO 48:263-268 (1973)). Analysis of the neutralization pattern affords an identification of serotype. (See Rotbart, 1995). Clearly, this is a cumbersome and painstaking process. Additionally, the supply of the antisera is limited or difficult to maintain. Problems in serotyping more recent isolates have been ascribed to pronounced intratypic antigenic variation (Meinick, Enteroviruses: polioviruses, coxsackie viruses, echoviruses, and newer enteroviruses. In Fields Virology (Fields et al., (Eds.) 3rd Ed., Lippincott-Raven Publishers, Philadelphia, 1996, pp. 655-712; Melnick et al., Bull. W.H.O. 63:453-550 (1985); Wigand et al., Arch. Ges. Virusforsch. 12:29-41 (1962); Wenner et al., Am J. Epidemiol. 85:240-249 (1967); Duncan, Arch. Ges. Virusforsch. 25:93-104 (1968)). This has been explained by pointing out that enteroviruses, being RNA viruses, undergo spontaneous mutation at a very high rate. This can lead to antigen drift, with the potential of producing antigenic variants such that a neutralization assay would produce a false negative result. For example, escape mutants in picornaviruses are discussed in detail in Mateu (Virus Res. 38:1-24 (1995)). For all these reasons there is a need to supplant neutralization assays for serotyping non-polio enteroviruses.
More recently assays based on nucleic acid detection have been developed. Probe hybridization assays directed either to RNA or to cDNA have been used to detect non-polio enteroviruses (Rotbart et al., Mol. Cell. Probes 2:65-73 (1988); Rotbart, J. Clin. Microbiol. 28:438-442 (1990); Chapman et al., J. Clin. Microbiol. 28: 843-850 (1990); Hyypia et al., J. Gen. Virol. 70:3261-3268 (1989); Olive et al. J. Gen. Virol. 71:2141-2147 (1990); Gilmaker et al., J. Med. Virol. 38:54-61 (1992); Yang et al., Virus Res. 24:277-296 (1992); Zoll et al., J. Clin. Microbiol. 30:160-165 (1992); Muir et al., J. Clin. Micro. 31:31-38 (1993); Drebot et al., J. Med. Virol. 44:340-347 (1994); Rotbart et al., J. Clin. Microbiol. 32:2590-2592 (1994)). In the absence of nucleic acid sequence information for the non-polio enteroviruses, most of these probes have targeted the highly conserved 5′ non-coding region of the viral genomes. Additionally, RNA probes directed to the VP1 capsid gene have been used on a limited basis to identify some of the CBs and a few closely related CAs (Cova et al., J. Med. Virol. 24:11-18 (1988); Alksnis et al., Mol. Cell. Probes 3:103-108 (1989); Petitjean et al., J. Clin. Microbiol. 28:307-311 (1990)). More recently, oligonucleotides having sequences based on the VP4-VP2 junction have been applied as diagnostic and epidemiologic tools (Drebot et al., J. Med. Virol. 44:340-347 (1994); Arola et al., J. Clin. Microbiol. 34:313-318 (1996); Kim et al., Arch. Virol. 142:853-860 (1997); Oberste et al., Virus Res. 58:35-43 (1998)). The sequences in these regions, however, do not always correlate with serotype (Kopecka et al., Virus Res. 38:125-136 (1995); Arola et at., J. Clin. Microbiol. 34:313-318 (1996)). Furthermore, sequences of only certain pritotpyes prototypes were available with which to compare and classify clinical samples (Arola et al., (1996)). A generic probe-based assay for nucleic acids in the presence of chaotropic agents is described in U.S. Pat. No. 5,726,012. An assay for a target nucleic acid sequence wherein two separate probes are hybridized to the same strand of a nucleic acid, and then joined, for example by a polymerase activity, is disclosed in U.S. Pat. No. 5,516,641.
Reverse transcription (RT) coupled with the polymerase chain reaction (PCR) (RT-PCR) has been developed using enterovirus universal primers or broadly selective primers. Such primers are intended to amplify nucleotide regions from a large number of enterovirus serotypes in one diagnosis. One set of primers (Rotbart, J. Clin. Microbiol. 28:438-442 (1990)) has been reported to amplify 60 of the 66 serotypes tested. (Among the nonreactive serotypes, two are a typical enteroviruses and may be reclassified.) A comparison of sequence identities of the various sets of universal primers with serotype sequences is given in Rotbart et al. (1995). Many of the universal primer sets are designed to amplify regions of the 5′ untranslated region of the genome (see, for example, Drebot et al. (1994); Diedrich et al., J. Med. Virol. 46:148-152 (1995); Arolaet al. (1996); Bailly et al., Virology 215:83-96 (1996); and U.S. Pat. No. 5,075,212 to Rotbart). A comparison of base sequences in coxsackievirus B5 was reported for isolates from three different outbreaks of disease, based on amplicons obtained using primers in the VP1/2A region of the genome (Kopecka et al., (1995)). Variations in sequence occurred even within the same outbreak, and somewhat greater variations were found among isolates from the different outbreaks, and between serotypes. International application WO 98/14611 discloses degenerate primers directed to the VP1 gene, which, when used in certain defined pairs, provide PCR amplification of enterovirus nucleic acids. Use of the specific primer pairs permits ascertaining whether a sample belongs to an enterovirus serotype, or to a small group of cognate serotypes, based on correlation of the pattern of the presence or absence of an amplicon with priming by the various primer pairs. This method does not rely on obtaining nucleotide sequences for accomplishing the serotyping.
Oberste et al. developed a database of homologous sequences for a portion of the VP2 gene of all 66 human enterovirus serotypes (Virus Res. 58:3545 (1998a)). They found that the sequences of many antigenic variants failed to cluster with their respective prototype strains as determined by serotyping. This finding suggested that the portion of VP2 examined may not prove to be useful for consistent molecular inference of serotype.
According to Holland et al. (J. Clin. Microbiol. 36:1588-1594 (1998)) neither cell culture growth, nor PCR can successfully type enterovirus infections. They report an alternative typing protocol based on polyacrylamide gel electrophoretic fingerprinting of whole virus radiolabeled proteins. However, the database of viral protein profiles contains data for less than one-third of the known EV serotypes. Therefore its general applicability remains unknown.
In the case of poliovirus, U.S. Pat. Nos. 5,585,477 and 5,691,134 to Kilpatrick disclose methods and oligonucleotide primers that are specific and sensitive for detecting all genotypes of poliovirus, as well as primers that are specific and sensitive for distinguishing the three serotypes of poliovirus, and methods for detecting poliovirus and/or distinguishing among the serotypes based on the use of the disclosed primers. Additionally WO 98/14611 discloses an extensive set of degenerate oligonucleotide primers for use in detecting the presence or absence of a non-polio enterovirus in a sample and to identify non-polio enterovirus serotypes. The primers are combined in pairs that detect various groupings of serotypes, and several amplification procedures are carried out in order to detect the presence of or absence of an amplicon in each case. A pooled grid of the results provides information useful in typing a non-polio enterovirus in a sample.
In summary, immunological methods for serotyping enteroviral infections are cumbersome and time consuming. They rely on an antigen-antibody reaction between antiserum pools established more than two decades ago, and whose supply may become limited. As explained, for example in Mateu (1995), antigen drift among RNA viruses such as the enteroviruses leads to a high probability that escape mutants will arise, and thereby escape not only serotyping, but perhaps detection as well. A second classical approach, cell culture coupled with whole animal host growth and use of antisera for typing, is extremely cumbersome, expensive, and labor-intensive. Modern molecular biological methods similarly have important deficiencies as currently implemented. Probe assays generally tend to lack sensitivity. Furthermore, a probe directed to a conserved region, such as the 5′ non-coding region of the non-polio enteroviruses, lacks specificity, and so cannot be readily applied in typing a viral infection. RT-PCR has been implemented as a generic enteroviral diagnostic assay. In general, these assays fail to implement serotype-specific detection, so that typing is not currently available using RT-PCR. Holland et al. (1998) state that all typing methods in use or then currently under development are limited by virtue of the large number of different enteroviral serotypes, and as a consequence, the need for virus-specific reagents that would discriminate among them.
For these reasons, there remains a need for a typing procedure that avoids the necessity of infecting live animals, animal tissue homogenates, or cell cultures. There further remains a need to implement a nucleic acid-based enteroviral typing procedure that optimizes the specificity required for a typing protocol. There additionally persists a need for a typing procedure that avoids a requirement for a plethora of reagents directed toward the specificity of the various serotypes. There still further remains the need for an enteroviral typing procedure that does not require extended periods of time or complicated procedures to carry out. Thus, there remains a need for an operationally elegant and efficient typing procedure that utilizes the specificity that resides, for example, in the VP1 region. The present invention recognizes these needs, and addresses them.